Specific Rab GTPase-activating proteins define the Shiga toxin and epidermal growth factor uptake pathways

Abstract

Rab family guanosine triphosphatases (GTPases) together with their regulators define specific pathways of membrane traffic within eukaryotic cells. In this study, we have investigated which Rab GTPase-activating proteins (GAPs) can interfere with the trafficking of Shiga toxin from the cell surface to the Golgi apparatus and studied transport of the epidermal growth factor (EGF) from the cell surface to endosomes. This screen identifies 6 (EVI5, RN-tre/USP6NL, TBC1D10A-C, and TBC1D17) of 39 predicted human Rab GAPs as specific regulators of Shiga toxin but not EGF uptake. We show that Rab43 is the target of RN-tre and is required for Shiga toxin uptake. In contrast, RabGAP-5, a Rab5 GAP, was unique among the GAPs tested and reduced the uptake of EGF but not Shiga toxin. These results suggest that Shiga toxin trafficking to the Golgi is a multistep process controlled by several Rab GAPs and their target Rabs and that this process is discrete from ligand-induced EGF receptor trafficking.

Figure 1.

Comparison of the Shiga toxin and EGF uptake pathways. Fluorescently labeled STxB (red) and EGF (green) were bound on ice to the surface of HeLa cells starved of serum overnight. Excess ligands were washed away, and the cells were incubated in complete growth medium at 37°C for the times indicated in the figure. The cells were then fixed and mounted without further processing. DNA was stained with DAPI (blue). Bar, 10 μm.

Figure 2.

Shiga toxin but not EGF uptake is Rab6 dependent. (A and B) HeLa cells were treated with control or Rab6 siRNA for 72 h and were either fixed and stained with antibodies to either Rab6 or Bicaudal-D1 (red) and TGN46 (green; A) or used for Shiga toxin (red) and EGF (green) uptake assays and costained for TGN46 (green) or EEA1 (red; B). (C) HeLa cells transfected with the GFP-tagged Rab5^Q79A dominant-active mutant (green) for 24 h were used for Shiga toxin and EGF (red) uptake assays or were stained for EEA1 and transferrin receptor (red). DNA was stained with DAPI (blue). Bar, 10 μm.

Figure 3.

TBC1D11/GAPCenA is a GAP for Rab4 and does not block Shiga toxin uptake. (A) HeLa cells transfected with GFP-tagged TBC1D11/GAPCenA (green) were used for Shiga toxin uptake assays (red). (B) The ability of Rab6 and Bicaudal-D1 (red) to localize to the Golgi was tested in cells expressing GFP-tagged TBC1D11/GAPCenA (green). (C) A schematic of TBC1D11/GAPCenA shows the putative phosphotyrosine-binding domain (PTB; blue), the TBC domain (red), and a C-terminal coiled-coil region (green). Full-length TBC1D11/GAPCenA and the C-terminal coiled-coil region identified by unbiased yeast two-hybrid library screen prey constructs were tested against the human Rabome bait construct library as described in Materials and methods. All conditions showed equal growth on nonselective media; the ability to grow on the selective media shown in the figure is an indicator of an interaction between the bait and prey. (D) GAP assays were performed using 0.5 pmol of recombinant TBC1D11/GAPCenA (closed bars) or a buffer control (open bars) and 100 pmol of the Rabs indicated in the figure. Two preparations of Rab6 were used: one from bacteria (Rab6), and the other a prenylated preparation produced in insect cells (Rab6^Sf9). Error bars represent SD. Bar, 10 μm.

Figure 4.

A subset of human Rab GAPs influence Shiga toxin transport. (A) Shiga toxin uptake assays were performed on HeLa cells transfected with a library of wild-type human Rab GAPs. Those Rab GAPs showing a block in Shiga toxin uptake were retested as catalytically inactive RA mutants. To measure the extent of Shiga toxin uptake, HeLa cells were transfected with the GFP-tagged wild-type and catalytically inactive mutant GAPs indicated in the figure. These cells were then fixed and stained for a Golgi marker. The percentage of untransfected and transfected cells in which STxB had reached the Golgi was counted for each condition, and the specific reduction in Shiga toxin transport was calculated by subtracting these two figures (transfected − untransfected cells). These numbers are plotted on the bar graph. The dotted red line indicates the cut-off chosen for consideration as a positive. Asterisks indicate that transport was the same as in control cells. (B) Images of the Rab GAPs showing a catalytic activity–dependent block or reduction in Shiga toxin uptake after 60 min. Shiga toxin is in red, transfected Rab GAPs are in green, and DNA is stained blue. (C) Cytotoxicity assays were performed using the Rab GAPs listed in the figure. The IC₅₀ of Shiga-like toxin 1 was measured in cells expressing either the wild type (WT) or the catalytically inactive (RA) mutant GAPs. These values were then corrected for transfection efficiency averaging 13.9–18.7%. An increase in the IC₅₀ for wild type compared with RA GAPs indicates reduced cytotoxicity, and this is plotted in the graph as the fold protection, with 1 (red dotted line) corresponding to no protection (n ≥ 3). The corrected IC₅₀ values for each condition are shown above each column, and the IC₅₀ of empty vector–transfected control cells was 3.5 ng/ml. Error bars represent SD. Bar, 10 μm.

Figure 5.

Rab GAPs altering Shiga toxin transport do not prevent EGF uptake. (A) EGF uptake assays were performed on HeLa cells transfected with a library of wild-type human Rab GAPs listed in the figure. The number of cells binding and then taking up EGF was counted for each condition. The graph shows cells either failing to bind (open bars) or taking up (closed bars) EGF. (B) EGF uptake assays were performed on HeLa cells transfected with the Rab GAPs capable of blocking Shiga toxin uptake and RabGAP-5. The initially bound EGF at 0 min of transport and the extent of EGF transport at 30 min are shown. EGF is in red, transfected Rab GAPs are in green, and DNA is stained blue. Bar, 10 μm.

Figure 6.

Discrete Rab GAPs define the Shiga toxin and EGF uptake pathways. (A and B) HeLa cells were transfected for 24 h with RabGAP-5, the catalytically inactive RabGAP-5^R165A mutant, or RN-tre. (A) EGF uptake assays were performed, and the initially bound EGF at 0 min of transport and extent of EGF transport at 30 min are shown in the figure. EGF is in red, and transfected Rab GAPs are in green. (B) The cells were fixed and stained for the transfected Rab GAPs (green) and EEA1 (red). (C and D) HeLa cells were transfected for 24 h with RN-tre, the catalytically inactive RN-tre^R150A mutant, or RabGAP-5. (C) Shiga toxin uptake assays were performed, and the initially bound Shiga toxin at 0 min of transport and extent of STxB transport to the Golgi at 60 min are shown. STxB is in red, and transfected Rab GAPs are in green. (D) The cells were fixed and stained for the transfected Rab GAPs (green) and GM130 (red). DNA is stained blue in all panels. Bar, 10 μm.

Figure 7.

Biochemical screening for the target Rabs of specific TBC domain proteins. To determine the specific activity toward a range of GTPases, 0.5 pmol RN-tre, RabGAP-5, TBC1D10B, and TBC1D17 were tested against 100 pmol of the Rab GTPases indicated using the procedures described in Materials and methods. All reactions were performed for 60 min. The basal GTP hydrolysis seen with a buffer control was subtracted for each GTPase, and the mean GTP hydrolysis in picomoles/hour is plotted in the figures. Error bars represent SD.

Figure 8.

Functional verification of the targets of RN-tre and RabGAP-5. (A–D) HeLa cells were transfected with siRNA duplexes to deplete all three isoforms of Rab5 (A and B) or Rab43 (C and D). (A and D) EGF uptake assays were performed, and the extent of EGF transport at 30 min is shown in the figure. EGF is in green, and EEA1 is in red. (B and C) Shiga toxin uptake assays were performed, and the extent of STxB transport to the Golgi at 30 min is shown. STxB is in red, and GM130 is in green. (E) HeLa cells transfected with GFP-tagged Rab43 (green) were fixed after 24 h and stained with antibodies to the Golgi marker TGN46 or the early endosome marker EEA1 (red). DNA is stained blue in all panels. Bar, 10 μm.